1-androstene-3, 11, 17-trione



2,874,169 1-ANDROSTENE-3,1l,17-TRIONE Samuel H. Eppstein, Galesburg, andPeter D. Meister,

Kalamazoo Township, Kalamazoo, County, Mich., and Adolph Wemtraub,Brooklyn, N. Y., assignors to The Upjohn Company, Kalamazoo, Mich, acorporation of Michigan No Drawing. Application November 19,1956 SerialN0. 622,830

3 'Claims. (Cl. 260-397.3)

This invention relates to new chemical-compounds,

1 ICC 2 was adjusted with a 25 percent sodium hydroxide somtion to a pHof 4.9 and sterilized for 45 minutes at twenty pounds steam pressure .ina fifty-gallon fermenter, The medium wascooled to 28Idegrees centigradeand inoculated with six percent by volume of a 24-hour growth fromspores of Septomqyxa a ffinis, A. T. C. C. 6737. The

medium was agitated with a sweep stirrer at 200 RIP-QM. and sterile airadmitted through a sp'arger at the rate of one liter per minute. After aperiod of nineteen hours, "twenty grams of allopregnane-3,l1,20-trionedissolved in 2000 milliliters of hot dipropylene glycol was added. The

"pH ofthe medium at this time was 4.9.. Fermentation was allowed tocontinue for a period *ofl24 hours at 1,5u-androstene-3,11,17-trione andl,5B-androstene-3,11,

17-trione, which compounds possess valuable anabolic and androgenicactivity.

This application is a continuation-in-part of application S. N. 493,302,filed March 9, 1955, now abandoned.

The compounds of this invention have the following structural formula:

The novel compounds of this invention can be produced by thefermentative l-dehydrogenation of allopregnane-3,1l,20-trione andpregnane-3,l1,20trione, respectively, with a fungus of the genusSeptomyxa, under aerobic conditions as disclosed in the copending priorapplication previously referred to.

As disclosed in the prior application, the operational conditions andreaction procedure and details of production can be those already knownin the art of steroid bioconversion as illustrated by U. S. Patent2,602,769, utilizing however the action of a species of fungus of thegenus Septomyxa. The genus Septomyxa belongs to the class ofDeuteromyces, Fungi Imperfecti, of the order Melanconi-ales, of thefamily Melaconiaceae. Among the species of the genus Septomyxa which areuseful in the conversion of allopregnane-3,11,20-trione to1,5a-androstene-3,1l,17-tri0ne and of pregnane-3,ll,20-trione to 1,518androstene 3,11,17 trione are Septomyxa afiinis (Sherb.) Wr., A. T. C.C. 6737, American Type Culture Collection, 2029 M Street, N. W.,Washington 6, D. C., Septomyxa aesculi, Septomyxa corni, Septomyxasalicina, and Septomyxa tulasnei.

As disclosed in the prior application, culture of the fungi for theproduction of l-dehydrogenation is in or on a medium favorable to thedevelopment of the fungi, employing conventional sources of assimilablecarbon and assimilable nitrogen; and using conventional sources ofmineral nutrients.

The following examples are illustrative of the production of the novelcompounds, and are not to be construed as limiting.

EXAMPLE 1 Preparation of 1,5ot-andr0stene-3JL17-tri0ne One hundredliters of a medium containing the following:

Cornsteep liquor (sixty percent solids) 2% by weight. Dextrose hydrate1% by welght. Lard oil (ten percent octadecanol) 80 milliliters.

which time the pH was 5.7. Analysis of an aliquot-of the medium showedthat all the substrate had been-converted. A major product wasidentified as the compound of this invention. The beer was heated todegrees centigrade for fifteen minutes, cooled, and filtered. Theseparated mycelium and filtrate were each extracted with methylenechloride and the extract washed with a two percent sodium bicarbonatesolution. This was followed by a wash with water, and the extract wasdried with sodium sulfate. The extract was concentrated to dryness,yielding a residue of 123.3 grams.

The residue was triturated with ether and then with a mixture of etherand Skellysolve B hexanes. Upon cooling, the solution yielded 12.2 gramsof crude crystals, M. P. 233.5 to 242 degrees centigrade. The crudecrystals were recrystallized from methanol and then from a mixture ofchloroform and Sellysolve B hexanes. A final recrystallization fromchloroform-ether gave 5.1 grams of pure crystals of1,5u-androstene-3,11,17-trione, M. P. 244.5 to 246.5 degrees centigrade,[a] =188 in CHCl (1.049 cone.)

x i; 228 my a 11,400. Infrared spectrum indicates absorption as follows:17-ketone, 1736 cm.- ll-ketone, 1701 cmr conjugated ketone, 1665 cm.- ;A-structure (C=C), 1600 emr The ultraviolet absorption maxima at 228mmconfirms the A -3-ketone system.

Analysis.Calculated for C H O C, 75.97; H, 8.05. Found: C, 75.20; H,8.06.

EXAMPLE 2 1,5,3-an'dr0stene-3J 1,1 7- trione Twelve liters of mediummade as in Example 1 was sterilized in a five-gallon fermenter at twentypounds steam pressure for sixty minutes. After cooling to 28 degreescentigrade, the medium was inoculated with five percent by volume of a77-hour growth from spores of Septomyxa afiinis, A. T. C. C. 6737.Sterile air was admitted through a sparger at the rate of one liter perminute, while the stirrer of the fermenter was adjusted to 200 R. P. M.After 24 hours for growth of the culture, 2.0 grams of pregnane-3,11,20-trione, dissolved in milliliters of hot propylene glycol, was added andthe fermentation continued for an additional period of 48 hours, atwhich time the pH of the beer was 7.2. After separation of the myceliumfrom the aqueous solution by filtration, extraction of each fraction wascarried out in the same manner as in Example 1 and the combinedmethylene chloride extracts concentrated until solvent-free. Theisolation followed the same procedure as in Example 1, yielding, in thiscase, 1,5Bandrostene- 3,11,17-trione. Recrystallization, following theprocedure 3 of Example 1, yielded purified product, M. P. 174.5 to 177.5degrees centigrade. Infrared spectrum indicates absorption as follows:17-ketone, 1740 cmr ll-ketone, 1700 cmr A -3-ketone, 1674 cmr- A-structure (C=C), 1605 cmr The ultraviolet absorption maxima at 224 m,confirms the A -3-ketone system. The a value was 8,500

1,5wandrostene 3,11,17 trione and 1,5;3-androstene- 3,11,17-trione ofthis invention exhibit valuable anabolic and androgenic activity and canbe administered in the form of tablets, capsules, syrups and the likefor oral use or in suitable conventional suspension media for injectionuse.

It is to be understood that the invention is not to be limited to theexact details of operation or exact compounds shown and described, asobvious modifications and equivalents will be apparent to one skilled inthe art, and the invention is therefore to be limited only by the scopeof the appended claims.

We claim: 1. A compound of the following structural formula:

UNITED STATES PATENTS Butenandt May 18, 1948 Murray July 8, 1953

1. A COMPOUND OF THE FOLLOWING STRUCTURAL FORMULA